Salvia miltiorrliza. The plant, Salvia miltiorrhiza (SM), has long been used in traditional Chinese medicine for treatment of cardiovascular and hepatic diseases. Extracts from the plant confer potent hepatoprotective activity both in vitro and in vivo. Hase et al., Planta Med. 63: 22-6 (1997). Magnesium lithospermate B may be one of the main active components of SM that protects the liver (Liu et al., Chung Kuo Chung Hsi I Chih Ho Tsa Chih 13: 352-3, 326 (1993)). SM also contains antioxidants that apparently aid membrane damage repair when treating viral myocarditis (Meng et al., Chung Kuo Chung Hsi I Chieh Ho Tsa Chih 12: 345-7, 324-5 (1992)). Patients suffering from chronic hepatitis B have responded to treatment with SM and/or Polyporus Umbellatus polysaccharidc (PUP) (Xiong, Chung Kuo Chung Hsi I Chieh Ho Tsa Chih 13: 33-5, 516-7 (1993)). Herbal extracts from SM also have demonstrated anti-HIV activity (U.S. Pat. No. 5,178,865) and anti-hepatitis activity (International PCT Application 98/24460; Chinese Patent Application Nos. 1,192,922 and 1,192,918). Antiviral agents active against herpes, polio, measles, varicella zoster, cytomegalovirus, DNA viruses and RNA viruses have been described which contain at least one crude drug from the root of Salvia miltiorrhiza Bunge (European Patent No. 568,001). Salvia extracts have also been prepared as anti-herpes virus agents (U.S. Pat. No. 5,411,733).
The SM plant has several components which may be extracted. Components of the root have been extracted initially with ethanol, followed by extraction with cold water (SM(1)) or with hot water (SM(2)). Both fractions extracted in water have shown antiviral activity.
Antiviral agents have been developed which target different points in a virus life cycle. For example, antiviral agents for treating retroviral infections have been developed which target retrovirus specific enzymes such as reverse transcriptase (RT) and integrase. Research continues to identify other antiviral agents to combat diseases such as herpes, hepatitis and influenza.
Antiviral Agents Against Retroviruses. Antiviral agents targeting the integrase protein include peptide inhibitors (U.S. Pat. No. 5,578,573), nucleic acid ligand inhibitors (U.S. Pat. Nos. 5,756,287 and 5,587,468), as well as compounds such as Equisetin (U.S. Pat. Nos. 5,759,842) and ermophilane sesquiterpenoids (U.S. Pat. No. 5,858,738). To date, of the numerous compounds that have already been identified and approved for marketing by the FDA for HIV, only RT and protease inhibitors have been approved.
Caffeic Acid as an Antiviral Agent. Caffeic acid can be isolated from the stems of Bougainvillea spectabillis Wild (Nyctaginaceae), which has been used as folk medicine against hepatitis (Chang et al., Anticancer Res. 14: 501-6 (1994)). Caffeic acid has been reported to inhibit xanthine oxidase, which is associated with several diseases, e.g., gout, hepatitis and tumors (Chan et al., Anticancer Res. 15: 703-7(1995); and Chang et al. (1994)). Caffeic acid oxidation product (KOP) inhibits herpes virus hominis type 1 and type 2 (Thiel et al., Acta Virol. 27: 200-8 (1983)). A caffeic acid tetramer and dipotassium and potassium-sodium salts of a caffeic acid tetramer glucoside possess anti-HIV activity according to Kashiwada et al., J. Nat. Prod. 58: 392-400 (1995). Commercially produced caffeic acid also has antiviral activity, as demonstrated using a RT assay for HIV (Kreis et al., Antiviral Res. 14: 323-37 (1990)). Caffeic acid phenethyl esters (CAPE) exert inhibitory activity on the integrase protein of HIV- 1 (Fesen et al., Proc. Natl Acad. Sci. USA 90: 2399-2403 (1993); Fesen et al., Biochem. Pharmacol. 48: 595-608 (1994); Burke et al., J. Med. Chem. 38: 4171-8 (1995); Mazumder et al., J. Med. Chem. 39: 2472-81 (1996)). However, the method used to prepare caffeic acid polymers influenced their HIV-1 and HIV-2 inhibitory activity (Nakashima et al., Chem. Pharm. Bull. (Tokyo) 40: 2102-5 (1992)). Caffeic acid, along with cinnamic acid and rosemarinic acid, have also been proposed for treating influenza virus because of their antioxidant activity (International PCT Application 98/30228).
Rosemarinic and Lithospermic Acids as Antiviral Agents. Rosemarinic acid is a dimer of caffeic acid. A dimer of rosemarinic acid is lithospermate B. Both rosemarinic acid and lithospermic acid have been identified in extracts of SM root (Kohda et al., Chem. Pharm. Bull. (Tokyo) 37: 1287-90 (1989)). Rosemarinic acid possesses anti-HIV activity (Arda et al., J. Nat. Prod. 60: 1170-3 (1997)) and is potentially a Herpes simplex type 1 (HSV-1) inhibitor (Dimitrova et al., Acta Microbiol. Bulg. 29: 65-72 (1993)). Rosemarinic acid also has been proposed to treat inflammatory diseases and disorders (U.S. Pat. No. 4,329,361).
Cinnamic Acid as an Antiviral Agent. Substituted cinnamic acid esters inhibit the infectious activity of influenza virus A/Hong Kong (H3N2) (Serkedjieva et al., J. Nat. Prod. 55: 294-302 (1992)); steryl esters of cinnamic acid derivatives have demonstrated antiviral activity in vitro against viruses belonging to Picornaviridae, Orthomyxoviridae, Paramyxoviridae and Herpesviridae ("Antiviral activity of cholesteryl esters of cinnamic acid derivatives," Z. Naturforsch 53: 883-7 (1998); and Conti et al., Antivir. Chem. Chemother. 9: 511-5 (1998)). Dehydrogenation polymers of substituted cinnamic acids have also been described as a HIV-1 therapeutic (U.S. Pat. Nos. 5,346,695 and 5,632,980).
Salvianolic Acid. Salvianolic acid has not been described in the literature as having antiviral properties. Several forms of salvianolic acid (e.g., salvianolic acid A, acetylsalvianolic acid ) have been described as having antioxidant properties (Lin et al., J Biochem. Pharmacol. 51: 1237-1241 (1996). Salvianolic acid has also been indicated for preventing liver injury and fibrosis, associated with its anti-lipid peroxidation actions (Hu et al., Acta Pharmacol. Sin. 18: 478-480 (1997)) and for use in treating coronary diseases (Japanese Patent 2,131,423). Additional forms of salvianolic acid described in the literature include those isolated from aqueous extracts of Salvia cavaleriei (e.g., salvianolic acids A, B, C H and I) (Zhang et al., Planta Med. 60: 70-72 (1994)) or from S. miltiorrhiza (e.g., salvianolic acid K, a caffeic acid trimer) (Kasimu et al., Chem. Pharm. Bull. 46: 500-504 (1998); and Tezuka et al., Chem Pharm. Bull. 46: 107-112 (1998)). Salvianolic acids F 2 and F 3 can be prepared synthetically as described by Dalla et al., Tetrahedron 55: 6923-6930 (1999); and Dalla et al., Tetrahedron Lett. 39: 8285-8286 (1998). Additional members of the Salvia family may be used as sources for obtaining Danshen, including: S. bowleyana, S. deserta, S. miltiorhiza var. miltiorhiza f. alba, S. paramiltiorhiza, S. paramiltiorhiza f. purpureo-rubra, S. przewalskii, S. prsewalskii var. mandarinorum, S. sinica f. purpurea, and S. trijuga) (Kasiumu et al., 1998). Methods of producing plants with elevated secondary metabolite levels of compounds such as salvianolic acid have also been described. See U.S. Pat. No. 5,869,340 (1999).
(3,4-dihydroxyphenyl) lactic acid. A principle component of SM is "Danshensu," chemically known as sodium .delta.-3,4-dihydroxyphenyl lactate (Fen et al., Acta Acad. Med. Primae Shanghai 10: 133-6 (1983); Zhao et al., Chin. Pharm. J. 29: 291-93 (1994)). It is an intermediate compound used in preparing rosemarinic acid in cell culture (Al-Sereiti et al., Indian J Exp. Biol. 37: 124-130 (1999); Bogucki et al., Can. J Chem. 75: 1783-94 (1997); and U.S. Pat. No. 5,011,775).
Treating virus-mediated diseases with antiviral agents can be quite expensive. For example, HIV-1 treatment using reverse transcriptase and protease inhibitors costs approximately $12,000-20,000 per patient per year. Given that the majority of HIV infected patients reside in developing nations, additional medications which are more economically attractive must be identified for the treatment of HIV and other viral diseases. The instant invention provides a new method of identifying, making and using novel compounds and compositions thereof for use as antiviral therapeutics.